Morphological subprofile analysis for bioactivity annotation of small molecules.

Fast prediction of the mode of action (MoA) for bioactive compounds would immensely foster bioactivity annotation in compound collections and may early on reveal off-targets in chemical biology research and drug discovery. Morphological profiling, e.g., using the Cell Painting assay, offers a fast, unbiased assessment of compound activity on various targets in one experiment. However, due to incomplete bioactivity annotation and unknown activities of reference compounds, prediction of bioactivity is not straightforward. Here we introduce the concept of subprofile analysis to map the MoA for both, reference and unexplored compounds. We defined MoA clusters and extracted cluster subprofiles that contain only a subset of morphological features. Subprofile analysis allows for the assignment of compounds to, currently, twelve targets or MoA. This approach enables rapid bioactivity annotation of compounds and will be extended to further clusters in the future.

Design and Synthesis of Metabolically Stable tRNA Synthetase Inhibitors Derived from Cladosporin.

Selective and specific inhibitors of Plasmodium falciparum lysyl-tRNA synthetase represent promising therapeutic antimalarial avenues. Cladosporin was identified as a potent P. falciparum lysyl-tRNA synthetase inhibitor, with an activity against parasite lysyl-tRNA synthetase >100-fold more potent than that of the activity registered against the human enzyme. Despite its compelling activity, cladosporin exhibits poor oral bioavailability; a critical requirement for antimalarial drugs. Thus, the quest to develop metabolically stable cladosporin-derived analogues, while retaining similar selectivity and potency to that of the natural compound, has begun. Chemogenomic profiling of a designed library allowed an entirely innovative structure-activity relationship study to be initiated; this shed light on structural evidence of a privileged scaffold with a unique activity against tRNA synthetases.

miRs-138 and -424 control palmitoylation-dependent CD95-mediated cell death by targeting acyl protein thioesterases 1 and 2 in CLL.

Resistance toward CD95-mediated apoptosis is a hallmark of many different malignancies, as it is known from primary chronic lymphocytic leukemia (CLL) cells. Previously, we could show that miR-138 and -424 are downregulated in CLL cells. Here, we identified 2 new target genes, namely acyl protein thioesterase (APT) 1 and 2, which are under control of both miRs and thereby significantly overexpressed in CLL cells. APTs are the only enzymes known to promote depalmitoylation. Indeed, membrane proteins are significantly less palmitoylated in CLL cells compared with normal B cells. We identified APTs to directly interact with CD95 to promote depalmitoylation, thus impairing apoptosis mediated through CD95. Specific inhibition of APTs by siRNAs, treatment with miRs-138/-424, and pharmacologic approaches restore CD95-mediated apoptosis in CLL cells and other cancer cells, pointing to an important regulatory role of APTs in CD95 apoptosis. The identification of the depalmitoylation reaction of CD95 by APTs as a microRNA (miRNA) target provides a novel molecular mechanism for how malignant cells escape from CD95-mediated apoptosis. Here, we introduce palmitoylation as a novel posttranslational modification in CLL, which might impact on localization, mobility, and function of molecules, survival signaling, and migration.

Omuralide and vibralactone: differences in the proteasome- ß-lactone-γ-lactam binding scaffold alter target preferences.

Despite their structural similarity, the natural products omuralide and vibralactone have different biological targets. While omuralide blocks the chymotryptic activity of the proteasome with an IC50 value of 47 nM, vibralactone does not have any effect at this protease up to a concentration of 1 mM. Activity-based protein profiling in HeLa cells revealed that the major targets of vibralactone are APT1 and APT2.

Characterization of a serine hydrolase targeted by acyl-protein thioesterase inhibitors in Toxoplasma gondii.

In eukaryotic organisms, cysteine palmitoylation is an important reversible modification that impacts protein targeting, folding, stability, and interactions with partners. Evidence suggests that protein palmitoylation contributes to key biological processes in Apicomplexa with the recent palmitome of the malaria parasite Plasmodium falciparum reporting over 400 substrates that are modified with palmitate by a broad range of protein S-acyl transferases. Dynamic palmitoylation cycles require the action of an acyl-protein thioesterase (APT) that cleaves palmitate from substrates and conveys reversibility to this posttranslational modification. In this work, we identified candidates for APT activity in Toxoplasma gondii. Treatment of parasites with low micromolar concentrations of ß-lactone- or triazole urea-based inhibitors that target human APT1 showed varied detrimental effects at multiple steps of the parasite lytic cycle. The use of an activity-based probe in combination with these inhibitors revealed the existence of several serine hydrolases that are targeted by APT1 inhibitors. The active serine hydrolase, TgASH1, identified as the homologue closest to human APT1 and APT2, was characterized further. Biochemical analysis of TgASH1 indicated that this enzyme cleaves substrates with a specificity similar to APTs, and homology modeling points toward an APT-like enzyme. TgASH1 is dispensable for parasite survival, which indicates that the severe effects observed with the ß-lactone inhibitors are caused by the inhibition of non-TgASH1 targets. Other ASH candidates for APT activity were functionally characterized, and one of them was found to be resistant to gene disruption due to the potential essential nature of the protein.

Design, synthesis and evaluation of polar head group containing 2-keto-oxazole inhibitors of FAAH.

2-α-Keto oxazoles containing polar head groups in their C5-side chains were designed as fatty acid amide hydrolase (FAAH) inhibitors. Variation in the spacer length resulted in submicromolar α-keto-oxazole FAAH inhibitor (IC(50)=436 nM) presenting electrostatic stabilizing interactions between its polar head group contained in the C5-side chain and the hydrophilic pocket of the enzyme.

Identification of acyl protein thioesterases 1 and 2 as the cellular targets of the Ras-signaling modulators palmostatin B and M.

Finding the target: activity-based proteomic profiling probes based on the depalmitoylation inhibitors palmostatin B and M have been synthesized and were found to target acyl protein thioesterase 1 (APT1) and 2 (APT2) in cells.

Development of highly potent inhibitors of the Ras-targeting human acyl protein thioesterases based on substrate similarity design.

A matter of common sense: a common recognition motif consisting of a negatively charged group five to six bonds away (red) from the (thio)ester functionality (green) and a positively charged tail group ten to twelve bonds away (blue) was identified in two native acyl protein thioesterase 1 (APT1) substrates. This similarity led to the design of potent inhibitors of the Ras-depalmitoylating enzyme APT1.

Small-molecule inhibition of APT1 affects Ras localization and signaling.

Cycles of depalmitoylation and repalmitoylation critically control the steady-state localization and function of various peripheral membrane proteins, such as Ras proto-oncogene products. Interference with acylation using small molecules is a strategy to modulate cellular localization--and thereby unregulated signaling--caused by palmitoylated Ras proteins. We present the knowledge-based development and characterization of a potent inhibitor of acyl protein thioesterase 1 (APT1), a bona fide depalmitoylating enzyme that is, so far, poorly characterized in cells. The inhibitor, palmostatin B, perturbs the cellular acylation cycle at the level of depalmitoylation and thereby causes a loss of the precise steady-state localization of palmitoylated Ras. As a consequence, palmostatin B induces partial phenotypic reversion in oncogenic HRasG12V-transformed fibroblasts. We identify APT1 as one of the thioesterases in the acylation cycle and show that this protein is a cellular target of the inhibitor.

A Pd-catalyzed heteroannulation approach to 2,3-disubstituted furo[3,2-c]coumarins.

The Pd-catalyzed cyclofunctionalization of 3-alkynyl-4-methoxycoumarins with aryl halides resulted in the selective formation of 3-arylfuro[3,2-c]coumarins in lieu of the expected regioisomeric 3-arylfuro[2,3-b]chromones. A mechanism involving the linear to angular rearrangement of a Pd-containing furan intermediate was proposed.